Glutathione Reductase
] Overview Glutathione reductase (GR) is a redox flavoenzyme found in a variety of organisms that catalyzes the reduction of oxidized, disulfide-containing glutathione dimer (GSSG) to the reduced monomer form (GR) through the use of reduced nicotinamide adenine dinucleotide phosphate (NADPH) as an electron donor. This helps the organism control oxidative stress as it regenerates GSH, which in turn can be used to reduce reactive oxygen and radical species (ROS) into a more benign form (ex. hydrogen peroxide to water). Metabolic Relevance ] Glutathione reductase is metabolically relevant in that it is integral to help keep available glutathione levels in check. Specifically, GR keeps a ratio of GSH to GSSG for the body to have available for use in antioxidant procedures. This is done through a process utilizing one-electron transfer through the FAD domain of the GR enzyme and the metabolic electron donor NADPH, reducing the GSSG disulfide and generating GSH.M. Deponte (2013)Glutathione catalysis and the reaction mechanisms of glutathione-dependent enzymes. Biochim Biophys Acta. 1830(5):3217-66. PubMed. As GSSG is a dimer of GSH molecules, the reaction produces two GSH for every GSSG reduced. Typically, the oxidized glutathione that is reduced produced via a detoxification mechanism in the body's antioxidant pathway. GR is found in all cells that GSH is present in, specifically mammalian cells, plant cells, and certain gram-negative bacteria. Recombinant Expression and Purification ] Recombinant GR is commercially available, generally from humans and expressed in E. Coli. GR is purified both recombinantly from E. Coli and directly from the cells (ex. human erythrocytes) of an organism in question. Due to purification procedures generally being both complicated and costly, purchasing commercially available GR is usually preferred. The expression of recombinant GR can be carried out utilizing pUB300 (a high copy plasmid based on pUC-18) and pJLA502 vector that has been cleaved with restriction enzymes and had the human GR Gene GSR ligated in alongside a plac promotor for expression regulation. The cells used are the SG5 strain of E. coli that produce little to no natural GR as to not contaminate the human form with the E. coli form.U. Bucheler, D. Werner, R. Schirmer. (1990) Random silent mutagenesis in the initial triplets of the coding region: a technique for adapting human glutathione reductase-encoding cDNA to expression in Escherichia coil. Gene 96, 271-276. PubMed Purification can also prove to be diffucult as GR must be separated from the similar redox protein Thioredoxin Reductase. The purification is generally carried out through the use of ammonium sulfate fractionation between 40-80% and subjected to two affinity columns: 2',5'-ADP sepharose and C8-ATPR sepharose. Both columns are eluted by means of an NADP+ gradient and fractions are analyzed by activity testing with GSSG, Absorbance at A280 and A460 for concentration, and gel electrophoresis for purity. A. Mata and M. Pinto. (1984) Purification by Affinity Chromatography of Glutathione Reductase (EC 1.6.4.2) from Escherichia coli and Characterization of such Enzyme. Z Naturforsch C. 39(9-10):908-15. PubMed Use of Recombinant Protein in Research Recombinant glutathione reductase can be used in a research setting to test the various activities of the protein on glutathione, glutathione-like substrates, and other biologically relvant reacive oxygen species.This can be done through spectrophotometrically following the dissapearance of NADPH in solution by tracking the absorbance at 340 nm. As the NADPH is used up, the absorbance should decrease yielding a slope that can be used to calculate enzyme activity through Beer's law and the concentration of enzyme used in solution.D. Worthington and M. Rosemeyer (1976) Eur J Biochem. 67(1):231-8. PubMed In addition to GSSG, GR has been found to have the ability to reduce other substrates such as the immune response oxidant Hypothiocyanous acid, and is thought to play more roles in redox biology than simply reducing GSSG.J.Chandler , D. Nichols, J. Nick ,R. Hondal , B. Day. (2013) Selective metabolism of hypothiocyanous acid by mammalian thioredoxin reductase promotes lung innate immunity and antioxidant defense. J Biol Chem. 288(25):18421–18428. PubMed GR can also be used as a means of measuring the activity of another related antioxidant enzyme, glutathione peroxidase (Gpx),as it provides an indirect way to measure the enzyme's activity through the usage of NADPH to reduce the oxidized glutathione produced by Gpx. This is especially helpful because there is currently no direct way to measure Gpx activity and its conversion of GSH to GSSG in the presence of an oxidant species. C. Weydert and J. Cullen. (2010) MEASUREMENT OF SUPEROXIDE DISMUTASE, CATALASE, AND GLUTATHIONE PEROXIDASE IN CULTURED CELLS AND TISSUE. Nat Protoc. 5(1): 51–66. PubMed References